Patrick Hsu

Multiplex Genome Engineering Using CRISPR/Cas Systems

Genome Editing Clustered regularly interspaced short palindromic repeats (CRISPR) function as part of an adaptive immune system in a range of prokaryotes: Invading phage and plasmid DNA is targeted for cleavage by complementary CRISPR RNAs (crRNAs) bound to a CRISPR-associated endonuclease (see the Perspective by van der Oost). Cong et al. (p. 819, published online 3 January) and Mali et al. (p. 823, published online 3 January) adapted this defense system to function as a genome editing tool in eukaryotic cells. A bacterial genome defense system is adapted to function as a genome-editing tool in mammalian cells. [Also see Perspective by van der Oost] Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.

Development and Applications of CRISPR-Cas9 for Genome Engineering

Recent advances in genome engineering technologies based on the CRISPR-associated RNA-guided endonuclease Cas9 are enabling the systematic interrogation of mammalian genome function. Analogous to the search function in modern word processors, Cas9 can be guided to specific locations within complex genomes by a short RNA search string. Using this system, DNA sequences within the endogenous genome and their functional outputs are now easily edited or modulated in virtually any organism of choice. Cas9-mediated genetic perturbation is simple and scalable, empowering researchers to elucidate the functional organization of the genome at the systems level and establish causal linkages between genetic variations and biological phenotypes. In this Review, we describe the development and applications of Cas9 for a variety of research or translational applications while highlighting challenges as well as future directions. Derived from a remarkable microbial defense system, Cas9 is driving innovative applications from basic biology to biotechnology and medicine.

Delivery and Specificity of CRISPR-Cas9 Genome Editing Technologies for Human Gene Therapy.

Genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated 9 (Cas9) technology is revolutionizing the study of gene function and likely will give rise to an entire new class of therapeutics for a wide range of diseases. Achieving this goal requires not only characterization of the technology for efficacy and specificity but also optimization of its delivery to the target cells for each disease indication. In this review we survey the various methods by which the CRISPR-Cas9 components have been delivered to cells and highlight some of the more clinically relevant approaches. Additionally, we discuss the methods available for assessing the specificity of Cas9 editing; an important safety consideration for development of the technology.

Methods for Optimizing CRISPR-Cas9 Genome Editing Specificity

Advances in the development of delivery, repair, and specificity strategies for the CRISPR-Cas9 genome engineering toolbox are helping researchers understand gene function with unprecedented precision and sensitivity. CRISPR-Cas9 also holds enormous therapeutic potential for the treatment of genetic disorders by directly correcting disease-causing mutations. Although the Cas9 protein has been shown to bind and cleave DNA at off-target sites, the field of Cas9 specificity is rapidly progressing, with marked improvements in guide RNA selection, protein and guide engineering, novel enzymes, and off-target detection methods. We review important challenges and breakthroughs in the field as a comprehensive practical guide to interested users of genome editing technologies, highlighting key tools and strategies for optimizing specificity. The genome editing community should now strive to standardize such methods for measuring and reporting off-target activity, while keeping in mind that the goal for specificity should be continued improvement and vigilance.

Twinfilin 2 Regulates Actin Filament Lengths in Cochlear Stereocilia

Inner ear sensory hair cells convert mechanical stimuli into electrical signals. This conversion happens in the exquisitely mechanosensitive hair bundle that protrudes from the cells apical surface. In mammals, cochlear hair bundles are composed of 50–100 actin-filled stereocilia, which are organized in three rows in a staircase manner. Stereocilia actin filaments are uniformly oriented with their barbed ends toward stereocilia tips. During development, the actin core of each stereocilium undergoes elongation due to addition of actin monomers to the barbed ends of the filaments. Here we show that in the mouse cochlea the barbed end capping protein twinfilin 2 is present at the tips of middle and short rows of stereocilia from postnatal day 5 (P5) onward, which correlates with a time period when these rows stop growing. The tall stereocilia rows, which do not display twinfilin 2 at their tips, continue to elongate between P5 and P15. When we expressed twinfilin 2 in LLC/PK1-CL4 (CL4) cells, we observed a reduction of espin-induced microvilli length, pointing to a potent function of twinfilin 2 in suppressing the elongation of actin filaments. Overexpression of twinfilin 2 in cochlear inner hair cells resulted in a significant reduction of stereocilia length. Our results suggest that twinfilin 2 plays a role in the regulation of stereocilia elongation by restricting excessive elongation of the shorter row stereocilia thereby maintaining the mature staircase architecture of cochlear hair bundles.

Dissecting neural function using targeted genome engineering technologies.

Designer DNA-binding proteins based on transcriptional activator-like effectors (TALEs) and zinc finger proteins (ZFPs) are easily tailored to recognize specific DNA sequences in a modular manner. They can be engineered to generate tools for targeted genome perturbation. Here, we review recent advances in these versatile technologies with a focus on designer nucleases for highly precise, efficient, and scarless gene modification. By generating double stranded breaks and stimulating cellular DNA repair pathways, TALE and ZF nucleases have the ability to modify the endogenous genome. We also discuss current applications of designer DNA-binding proteins in synthetic biology and disease modeling, novel effector domains for genetic and epigenetic regulation, and finally perspectives on using customizable DNA-binding proteins for interrogating neural function.

RNA-Guided Genome Editing of Mammalian Cells

The microbial CRISPR-Cas adaptive immune system can be harnessed to facilitate genome editing in eukaryotic cells (Cong L et al., Science 339, 819-823, 2013; Mali P et al., Science 339, 823-826, 2013). Here we describe a protocol for the use of the RNA-guided Cas9 nuclease from the Streptococcus pyogenes type II CRISPR system to achieve specific, scalable, and cost-efficient genome editing in mammalian cells.

Pairwise library screen systematically interrogates Staphylococcus aureus Cas9 specificity in human cells

Therapeutic genome editing with Staphylococcus aureus Cas9 (SaCas9) requires a rigorous understanding of its potential off-target activity in the human genome. Here we report a high-throughput screening approach to measure SaCas9 genome editing variation in human cells across a large repertoire of 88,692 single guide RNAs (sgRNAs) paired with matched or mismatched target sites in a synthetic cassette. We incorporate randomized barcodes that enable whitelisting of correctly synthesized molecules for further downstream analysis, in order to circumvent the limitation of oligonucleotide synthesis errors. We find SaCas9 sgRNAs with 21-mer or 22-mer spacer sequences are generally more active, although high efficiency 20-mer spacers are markedly less tolerant of mismatches. Using this dataset, we developed an SaCas9 specificity model that performs robustly in ranking off-target sites. The barcoded pairwise library screen enabled high-fidelity recovery of guide-target relationships, providing a scalable framework for the investigation of CRISPR enzyme properties and general nucleic acid interactions.A rigorous understanding of off-target effects is necessary for SaCas9 to be used in therapeutic genome editing. Here the authors measure SaCas9 mismatch tolerance across a pairwise library screen of 88,000 guides and targets in human cells and develop a model which ranks off-target sites.

Publisher Correction: Pairwise library screen systematically interrogates Staphylococcus aureus Cas9 specificity in human cells

The original HTML version of this Article incorrectly listed an affiliation of Josh Tycko as ‘Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA’, instead of the correct ‘Present address: Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA’. It also incorrectly listed an affiliation of this author as ‘Present address: Arrakis Therapeutics, 35 Gatehouse Dr., Waltham, MA, 02451, USA’.The original HTML version incorrectly listed an affiliation of Luis A. Barrera as ‘Present address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, 06511, USA’, instead of the correct ‘Present address: Arrakis Therapeutics, 35 Gatehouse Dr., Waltham, MA 02451, USA’.Finally, the original HTML version incorrectly omitted an affiliation of Nicholas C. Huston: ‘Present address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511, USA’.This has been corrected in the HTML version of the Article. The PDF version was correct from the time of publication.

317. Screening S. Aureus CRISPR-Cas9 Paired-Guide RNAs for Efficient Targeted Deletion in Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a recessive X-linked neuromuscular disorder that results in progressive muscle degeneration and premature death. Most patients have exonic deletions in the dystrophin gene that result in a frameshift and nonfunctional protein. In contrast, Becker muscular dystrophy (BMD) patients carry a range of exonic deletions in dystrophin that do not disrupt the reading frame, leading to a much milder disease phenotype. Thus, multiplex CRISPR/Cas9 targeted deletions that restore the reading frame could convert DMD genotypes into BMD-like genotypes and potentially treat this disease. Previously, this strategy has been demonstrated in vitro with zinc finger nucleases, TALENs, and S. pyogenes Cas9 leading to restoration of dystrophin expression in DMD patient myoblasts. However, these genome-editing enzymes are limited by the difficulty of delivering large transgenes with viral vectors in vivo. Alternatively, the smaller Cas9 ortholog from S. aureus (SaCas9) can be packaged with paired sgRNAs in an all-in-one AAV vector for in vivo gene therapy. Recently, three groups have demonstrated that AAV-SaCas9 can mediate targeted deletions in mdx mice and restore dystrophin expression. As a first step towards developing a genome editing therapeutic for DMD, we conducted an efficiency screen to identify highly active paired sgRNAs for targeted deletion of exon 51 of the dystrophin gene. First, a sensitive, digital droplet PCR assay to quantify exon 51 deletion was validated with DMD patient samples. An initial pilot study of 15 sgRNAs identified a pair (01+09) that mediated 18% Exon 51 deletion in HEK293T cells three days post-transfection. Next, from the set of 10,553 21-nt SaCas9 guides targeting human DMD introns 50 and 51, we selected 53 sgRNAs (675 pairs) that met the following filtering requirements: an endogenous 5’ G, a 3’ T in the NNGRR(T) PAM, cross-reactivity with the non-human primate (NHP) genome, and no off-by-1 or off-by-2 mismatch sites in the human genome. These filters were selected to improve U6 promoter expression, improve SaCas9 cleavage efficiency, enable pre-clinical animal model studies, and minimize off-target editing concerns, respectively. Notably, the NHP cross-reactivity requirement skewed the targeted deletion lengths towards larger sizes, all greater than 12.4kb. In order to test smaller deletions, an additional 174 pairs of human-only sgRNAs were designed for deletion lengths of 0.8-14kb. From the 850 guide pairs screened, 78 pairs with a deletion efficiency Z-score >1.5 were selected for follow-up validation. Interestingly, a few individual sgRNAs consistently appeared among the top performing pairs, regardless of whether they were paired with a generally less effective partner sgRNA. The top hit (guides 68+84), a human-only guide pair with a deletion size of 2.3kb, demonstrated a reproducible deletion efficiency of 32%. The 6 best human-only guide pairs and the 6 best NHP cross-reactive guide pairs have been cloned together with SaCas9 in all-in-one AAV vectors for testing in ex vivo and in vivo models of DMD.